Which of the following best describes how a bacterium is determined to be Gram

A Inherent Gram-Negative Resistance

Gram-negative bacteria are inherently resistant to macrolide antibiotics, presumably due to the sizes of the molecules (molecular weights of > 600) and their hydrophobicities. Studies (Farmer et al., 1992) indicate that macrolides cannot gain access into gram-negative bacteria via porins and that the new analog azithromycin, which has improved activity against gram-negative bacteria, may be gaining access via a self-promoted access pathway. Thus the polycationic nature of azithromycin enables it to competitively displace divalent cations from lipopolysaccharides in gram negative outer membrane, thereby increasing its own permeability. This observation may constitute a basis for the design of newer macrolides with improved activity against gram-negative bacteria. However, its validity was challenged by the observations of Vaara (1993), who found no cross-resistance between polymyxin, a compound which utilizes a selfpromoted penetration through the outer membrane, and azithromycin.

Etiology and Pathophysiology

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Gram-negative bacteria are the most common primary pathogens:

Often, these organisms are part of the normal flora, but they may become opportunistic.

Commonly isolated Gram-negative organisms include Pseudomonas, Klebsiella, Proteus, Salmonella, Providencia, Escherichia, Morganella, Aeromonas, and Citrobacter.

Occasionally, Gram-positive organisms (e.g., Streptococcus, Corynebacteria) are the primary organisms, or are found concurrently with Gram-negative bacteria.

Often, anaerobic organisms are found in conjunction with Gram-negative bacteria.

Anaerobic organisms include Bacteroides, Fusobacterium, and Clostridium.

Hypovitaminosis A is often a predisposing factor.

Chronic Tonsillitis.

AGNB can be involved in acute tonsillitis, chronic tonsillitis, and their complications, including internal jugular vein thrombophlebitis. Evidence of pathophysiologic features of anaerobes in nonstreptococcal tonsillitis includes the following: reduction of fever and clinical symptoms in patients treated with metronidazole compared with untreated children29; a detectable immune response against AGNB in patients with tonsillitis, peritonsillar cellulitis or abscess, and infectious mononucleosis30; isolation of AGNB from the cores of tonsils of children with recurrent tonsillitis23 and peritonsillar and retropharyngeal abscesses31; and isolation of aerobic and anaerobic β-lactamase–producing organisms from the tonsils of more than 75% of children with recurrent streptococcal tonsillitis.32 The ability to measure β-lactamase activity in the core of tonsils and patients' responses to agents effective against β-lactamase–producing bacteria (i.e., clindamycin or amoxicillin plus clavulanic acid) support the role of AGNB in children in whom penicillin has failed to eradicate streptococcal tonsillitis.32

Microbiologic Tests

Publisher Summary

Gram-negative bacteria possess a number of cell surface glycans that have been shown to play an important role in the biosynthesis and regulation of the cell wall of pathogenic Gram-negative bacteria. These glycans include lipopolysaccharides (LPSs), lipo-oligosaccharides (LOSs), capsular polysaccharides (CPSs), and N- and O-glycoproteins. This chapter discusses the role of surface carbohydrate structures in the ability of several invasive Gram-negative pathogens to interact with host cells, and the role of microbial glycosylation systems in host cell invasion of bacteria are compared. In some cases, the communication between the microbial pathogen and its host is due to recognition of these cell surface glycomolecules, thereby resulting in the ability of these organisms potentially to cause a multitude of infections and disease. In certain species, such as Shigella and Pseudomonas spp., the LPS core oligosaccharide is required for invasion.

1.1.1 Mortality Rate Due to Gram-negative Bacteria

Gram-negative bacteria manifest high level resistance to most classes of antibiotics (Zabawa et al., 2016). The infections caused by multidrug resistant (MDR) gram-negative organisms are increasing in hospitals, particularly in Intensive Care Unit (ICU) and are associated with higher costs, increased morbidity, and lead to high mortality rates (Chelazzi et al., 2015). Several factors that contribute to the increased risk of infection in ICU patients, includes greater severity of illness, overuse of existing antimicrobial agents, underlying conditions, exposure to multiple invasive devices and procedures, increased patient contact with health care personnel, and crowding of patients in a small specialized area (Ivady et al., 2015). Gram-negative bacteria, particularly Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumonia are causing health problems and especially to the hospitalized patients. There is an increasing demand to come up with alternative therapeutics that target MDR gram-negative infections (MacVane et al., 2015).

The rates of MDR gram-negative bacteria across the world generally suggest an increasing resistance towards the southeast of Europe, Latin America and Asia Pacific and lower resistance in the northwest of Europe, United States, and Canada (Curcio, 2014). In 2010, data from US National Healthcare Safety Network emphasized that gram-negative bacteria account for >30% of hospital-acquired infections, and in ICUs they represent for about 70% of these infections; similar data have reported from different parts of the world (Peleg and Hooper, 2010). In 2010, a systematic review included data from 47 countries and estimated that 13.5 million cases of typhoid fever occurred globally and around 200–300 cases of Salmonella typhi are reported each year in the United States (Bula Rudas et al., 2015). One of the effective strategies to combat MDR organisms is the development of novel antimicrobial agents (Cerceo et al., 2016). Fighting MDR bacterial infections with edible plants represents an attractive strategy (Dzotam et al., 2016).

Abstract

Gram-negative bacteria possess a complex cell envelope containing an outer membrane. The outer membrane is an asymmetric lipid bilayer that has an inner leaflet containing glycerophospholipids and, in almost all Gram-negative bacterial species, an outer leaflet whose major component is lipopolysaccharide (LPS), an amphiphilic glycolipid that is unique to Gram-negative bacteria. Lipopolysaccharide (LPS) is the major surface membrane component present in almost all Gram-negative bacteria and it is essential to both the form and function of the outer membrane. The distinctive structural properties of LPS molecules are crucial for the protective barrier properties of the outer membrane. Modifications of the general LPS structure also play important roles in host-pathogen interactions. LPS, a pathogen-associated molecular pattern, represents one of the conserved microbial structures responsible for activation of the innate immune system. In Gram-negative sepsis, LPS molecules released from the bacterial surface may stimulate an inflammatory response, which, if unchecked, can progress to the often-fatal syndrome known as septic shock. The involvement of LPS in this process is the origin of its “endotoxin” name, and these biological effects have inspired a substantial part of LPS research. The complex structures of LPS molecules also provide fascinating research topics in the areas of synthesis, export of macromolecules, LPS modification, and membrane biogenesis. The broad-spectrum of LPS research is reflected in the activities of the International Endotoxin and Innate Immunity Society (IEIIS) (http://www.ieiis.org/).

11.6.5 Gram-negative bacilli

GNB comprised 12% of all pathogens in PJI in a large retrospective cohort-study [15]; in this study 45% of such infections were polymicrobial with multiple GNB. Enterobacteriaceae, such as E. coli, Klebsiella, Proteus, and Enterobacter species are the most frequently encountered pathogens, followed by the nonfermentative Gram-negative rod Pseudomonas aeruginosa [15,27,59].

First choice treatment for enterobacteriaceae is an active beta-lactam antibiotic, such as a third-generation cephalosporin (ceftriaxone, cefotaxime), or, for more resistant isolates, a carbapenem. First-generation cephalosporins have a very limited spectrum of activity against enterobacteriaceae. Depending on local epidemiology, a second-generation cephalosporin may be used, although resistance is more common than against third-generation agents (first and second-generation cephalosporins do exhibit excellent activity against streptococci and methicillin-susceptible S. aureus, and are therefore good choices for peri-operative prophylaxis). Alternatively ciprofloxacin or cotrimoxazole could be used, but preferably these drugs are reserved for subsequent oral treatment. Therapy including ciprofloxacin is associated with higher cure rates in PJI caused by GNB [15,59]; if the pathogen is susceptible, a switch should therefore be made to (oral) ciprofloxacin after initial intravenous treatment. An oral alternative may be cotrimoxazole. There is currently no evidence to support combination therapy in the treatment of PJI caused by enterobacteriaceae.

Initial treatment of P. aeruginosa PJI is with an antipseudomonal beta-lactam, such as ceftazidime, piperacillin-tazobactam, or a carbapenem (meropenem, imipenem, doripenem, but not ertapenem). If susceptible, the treatment should be switched to oral ciprofloxacin. Ciprofloxacin is the only antibiotic associated with higher cure rates after a DAIR-procedure for P. aeruginosa. Furthermore, it is the fluoroquinolones (of which ciprofloxacin is the most active compound) that are the only oral antibiotics available for treatment of P. aeruginosa. Particular care should therefore be taken not to induce resistance to the drug. Experts are divided on whether combination therapy with aminoglycosides is indicated for P. aeruginosa PJI; its use is in any case not supported by evidence.

Lipopolysaccharide

Gram-negative bacteria have a cell envelope containing two membranes, the outer membrane is characterized by the presence of lipopolysaccharide in the outer leaflet of the bilayer structure. The lipopolysaccharide is involved in several aspects of pathogenicity. It serves as the hydrophobic anchor of Gram-negative bacteria. Lipopolysaccharide is a complex polymer of four parts. Outside of the cell there is a polysaccharide of variable structure known as O-antigen which carries several antigenic determinants. This is attached to a core polysaccharide of two parts, an outer core and a backbone. The cores vary between different bacteria. The backbone is connected to a glycolipid called lipid A, through a short link composed of 3-deoxy-d-mannooctulosonic acid. Lipid A consists of disaccharides of glucosamines that are highly substituted with phosphate, fatty acid, and 3-deoxy-d-man- nooctulosonic acid. The amino groups are substituted exclusively by 3-hydroxymyristate whereas the remaining hydroxyl groups are acylated with C12, C14, and C16 saturated fatty acids and 3-myristoxymyristate. There is microheterogeneity in bacteria with respect to fatty acids that are present in lipopolysaccharides.

Gram-Negative Organisms

Gram-negative organisms are a particularly important cause of nosocomial bloodstream infections, pneumonia, and meningitis because they generally cause severe disease. Escherichia coli is the most common gram-negative pathogen. Other gram-negative organisms responsible for HAI are Klebsiella, Pseudomonas, Enterobacter, Acinetobacter, Serratia, Haemophilus, and Salmonella spp. (see Box 40-3).

The attributable mortality is much higher for gram-negative infections than for gram-positive infections. Stoll et al (2002a) reported that infants with gram-negative infections had a 3.5-fold higher risk of death. Karlowicz et al (2000) found that gram-negative infections were associated with fulminant death within 48 hours of a positive blood culture result in 69% of cases. Pseudomonas spp. appear to be particularly virulent, causing death in 42% to 75% of infected neonates (Karlowicz et al, 2000; Leigh et al, 1995; Stoll et al, 2002a). A more recent study by Makhoul et al (2005) and colleagues in Israel confirms that gram-negative infections are associated with a substantially higher mortality.